Stable compositions for nucleic acid amplification and sequencing

ABSTRACT

The present invention is directed to compositions comprising mixtures of reagents, including thermostable enzymes (e.g., thermostable DNA polymerases), buffers, cofactors and other components, suitable for immediate use in nucleic acid amplification or sequencing techniques without dilution or addition of further components. The compositions contain no stablizing agents (e.g., glycerol or serum albumin) and unexpectedly maintain activity for extended periods of time upon storage at temperatures above freezing. These compositions are useful, alone or in the form of kits, for nucleic acid amplification (e.g., by the Polymerase Chain Reaction) and sequencing (e.g., by dideoxy or “Sanger” sequencing), or for any procedure utilizing thermostable DNA polymerases in a variety of medical, forensic and agricultural applications. In particular, the compositions and methods are useful for amplifying and sequencing nucleic acid molecules that are larger than about 7 kilobases in size.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/801,720, filed Feb. 14, 1997, which is a continuation-in-part of U.S.application Ser. No. 08/689,815, filed Aug. 14, 1996 (now abandoned),the contents of both of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

This invention is in the fields of molecular and cellular biology. Theinvention is particularly directed to reagent compositions for use intechniques whereby nucleic acids (DNA or RNA) are amplified orsequenced, and to methods of amplifying and sequencing long nucleic acidmolecules, especially via the polymerase chain reaction (PCR).

BACKGROUND OF THE INVENTION DNA Polymerases

During growth and reproduction of viruses and cellular organisms, theDNA containing the parental genetic information must be faithfullycopied and passed on to the progeny. This highly regulated process ofDNA replication is carried out in vivo by a complex of enzymes andassociated proteins and cofactors (Kornberg, A., Science 131:1503-1508,1959; I-Ebner, U., and Alberts, B. M., Nature 285:300-305, 1980). Theprimary enzymes taking part in this process are the DNA polymerases,which catalyze the addition of deoxynucleoside triphosphate (dNTP) basesinto the newly forming DNA strands. Together with other enzymes (e.g.,helicases, ligases and ATPases), the DNA polymerases thus ensure rapidand relatively faithful replication of DNA in preparation forproliferation in prokaryotes, eukaryotes and viruses.

DNA polymerases are also used to manipulate DNA in vitro in a variety ofmolecular genetic techniques. These enzymes have proven useful not onlyfor in vitro DNA synthesis, but also for determining the nucleotidesequence (i.e., “sequencing”) of DNA fragments or genes. This latterapplication relies on the fact that, in addition to an activity whichadds dNTPs to DNA in the 5′ to 3′ direction (i.e., “polymerase”activity), many DNA polymerases also possess activities which removedNTPs in the 5′ to 3′ and/or the 3′ to 5′ direction (i.e., “exonuclease”activity). DNA polymerases can also be used for sequencing via theirincorporation of labeled chain-terminating agents such asdideoxynucleoside triphosphates (See U.S. Pat. Nos. 4,962,020;5,173,411; and 5,498,523). Thus the same enzyme, e.g., DNA polymerases Iand III from the bacterium Escherichia coli, DNA polymerase γ fromanimal cells or DNA polymerase from bacteriophage T7 (See U.S. Pat. No.4,795,699), may be used in vitro both for DNA synthesis, involving theelongation of the DNA strand, and for DNA sequencing, involving eitherthe synthesis or the digestion of the DNA strand.

The dual activity of certain DNA polymerases is, however, a drawback forsome in vitro applications. For example, the in vitro synthesis of anintact copy of a DNA fragment by the polymerase activity, an elongationprocess which proceeds in a 5′ to 3′ direction along the template DNAstrand, is jeopardized by the exonuclease activities which maysimultaneously or subsequently degrade the newly formed DNA. To overcomethis technical problem, a fragment of E. coli DNA polymerase I lackingthe 5′ to 3′ exonuclease activity (named the “Klenow fragment” after itsdiscoverer) is often employed for in vitro DNA synthesis. The Klenowfragment provides for in vitro DNA synthesis at approximately the samerate as intact E. coli DNA polymerase I, but the newly synthesized DNAmolecules are less subject to enzymatic degradation and arecorrespondingly more stable.

Unfortunately, the error rate (i.e., the rate at which incorrect dNTPsare incorporated into the new DNA strand by the enzyme) is somewhathigher for the Klenow fragment than for many other commonly used DNApolymerases, including E. coli DNA polymerases I and II and thepolymerases from bacteriophages T4 and T7 (Kunkel, T. A., et al., J.Biol. Chem. 259:1539-1545, 1984; Tindall, K. R., and Kunkel, T. A.,Biochemistry 27:6008-6013, 1988; Mattila, P., et al., Nucl. Acids Res.19:4967-4973, 1991). Thus, until recently the rates of synthesis,degradation and error had to be weighed together when choosing which DNApolymerase to use for in vitro DNA synthesis.

DNA Sequencing

In general, two techniques have been traditionally used to sequencenucleic acids. In the first method, termed “Maxam and Gilbertsequencing” after its co-developers (Maxam, A. M. and Gilbert, W., Proc.Natl. Acad. Sci. USA 74:560-564, 1977), DNA is radiolabeled, dividedinto four samples and treated with chemicals that selectively destroyspecific nucleotide bases in the DNA and cleave the molecule at thesites of damage. By separating the resultant fragments into discretebands by gel electrophoresis and exposing the gel to X-ray film, thesequence of the original DNA molecule can be read from the film. Thistechnique has been used to determine the sequences of certain complexDNA molecules, including the primate virus SV40 (Fiers, W., et al.,Nature 273:113-120, 1978; Reddy, V. B., et al., Science 200:494-502,1978) and the bacterial plasmid pBR322 (Sutcliffe, G., Cold SpringHarbor Symp. Quant. Biol. 43:77-90, 1979).

An alternative technique for sequencing, named “Sanger sequencing” afterits developer (Sanger, F., and Coulson, A. R., J. Mol. Biol. 94:444-448,1975), is more commonly employed. This method uses the DNA-synthesizingactivity of DNA polymerases which, when combined with mixtures ofreaction-terminating dideoxynucleoside triphosphates (Sanger, F., etal., Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977) and a short primer(either of which may be detectably labeled), gives rise to a series ofnewly synthesized DNA fragments specifically terminated at one of thefour dideoxy bases. These fragments are then resolved by gelelectrophoresis and the sequence determined as described for Maxam andGilbert sequencing above. By carrying out four separate reactions (oncewith each ddNTP), the sequences of even fairly complex DNA molecules mayrapidly be determined (Sanger, F., et al., Nature 265:678-695, 1977;Barnes, W., Meth. Enzymol. 152:538-556, 1987). While Sanger sequencingusually employs E. coli or T7 DNA polymerase (U.S. Pat. No. 4,795,699),recent modifications of this technique using T7 polymerase mutants allowsequencing to be accomplished using a single sequencing reactioncontaining all four chain-terminating ddNTPs at different concentrations(U.S. Pat. Nos. 4,962,020 and 5,173,411). Further modifications to thetechnique, to reduce or eliminate the buildup of reaction-poisoningpyrophosphate in the reaction mixtures, have also been described (U.S.Pat. No. 5,498,523).

The Polymerase Chain Reaction

Soon after their identification and characterization, it was recognizedthat the activities of the various enzymes and cofactors involved in DNAsynthesis could be exploited in vitro to dramatically increase theconcentration of, or “amplify,” one or more selected DNA sequences. Formany medical, diagnostic and forensic applications, amplification of aparticular DNA sequence is essential to allow its detection in, orisolation from, a sample in which it is present in very low amounts.More recently, in vitro amplification of specific genes has providedpowerful and less costly means to facilitate the production oftherapeutic proteins by molecular biological techniques, and may haveapplications in genetic therapy as well.

While a variety of nucleic acid amplification processes has beendescribed, the most commonly employed is the Polymerase Chain Reaction(PCR) technique disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. Inthis process, a sample containing the nucleic acid sequence to beamplified (the “target sequence”) is first heated to denature orseparate the two strands of the nucleic acid. The sample is then cooledand mixed with specific oligonucleotide primers which hybridize to thetarget sequence. Following this hybridization, DNA polymerase in abuffered aqueous solution is added to the sample, along with a mixtureof the dNTPs that are linked by the polymerase to the replicatingnucleic acid strand. After allowing polymerization to proceed tocompletion, the products are again heat-denatured, subjected to anotherround of primer hybridization and polymerase replication, and thisprocess repeated any number of times. Since each nucleic acid product ofa given cycle of this process serves as a template for production of twonew nucleic acid molecules (one from each parent strand), the PCRprocess results in an exponential increase in the concentration of thetarget sequence. Thus, in a well-controlled, high-fidelity PCR process,as few as 20 cycles can result in an over one million-fold amplificationof the target nucleic acid sequence (See U.S. Pat. Nos. 4,683,195 and4,683,202).

Thermostable DNA Polymerases

Overview

Initially, the DNA polymerases of choice for use in DNA sequencing orPCR were E. coli DNA polymerase I, the Klenow fragment, or T4 or T7polymerases owing to their ease of isolation and well-characterizedactivities. However, the use of these enzymes necessitated theiraddition prior to the start of each sequencing or PCR cycle, due totheir thermolability at the temperatures used to denature the DNAstrands in the initial steps of the processes (typically 70° to 95° C.)(Saiki, R. K., et al., Science 230:1350-1354, 1985; Mullis, K. B., andFaloona, F. A., Meth. Enzymol. 155:335-350, 1987; U.S. Pat. No.4,795,699). This need for the addition of fresh enzyme at the beginningof each cycle increased the amount of time required for these processes,and increased the risk of operator error and contamination of thesamples during reagent introduction, often leading to undesirableresults.

These difficulties were partially overcome by the use of thermo stableDNA polymerases in the PCR process (Saiki, R. K., et al., Science239:487-491, 1988). The thermostable DNA polymerase most commonly usedin PCR is Taq polymerase, isolated from the thermophilic bacteriumThermus aquaticus (Saiki et al., 1988, Id.; U.S. Pat. Nos. 4,889,818 and4,965,188). Taq polymerase functions optimally at temperatures of 70-80°C., and is able to maintain substantial activity upon repeated exposureto temperatures of 92°-95° C. as are often used in the initial steps ofPCR (Gelfand, D. H., and White, T. J., in: PCR Protocols: A Guide toMethods and Applications, Innis, M. A., et al., eds., Academic Press,pp. 129-141, 1989; Bej, A. K., and Mahbubani, M. H., in: PCR Technology:Current Innovations, Griffin, H. G., and Griffin, A. M., eds., CRCPress, pp. 219-237, 1994).

The use of Taq polymerase in PCR eliminated the need to add fresh enzymeto the reaction mix prior to each PCR cycle. Instead, a quantity of Taqpolymerase sufficient to catalyze DNA polymerization over the desirednumber of cycles can be mixed with the other components prior to theinitiation of the first PCR cycle, and the enzyme continues to functionthroughout the repetitive cycles of increased and decreasedtemperatures. The use of Taq polymerase has also facilitated theautomation of the PCR process (Gelfand and White, Id), thereby at oncedramatically reducing time constraints and the risks of operator errorand sample contamination that are problematic with thermolabilepolymerases. Currently, most PCR amplification of nucleic acids forindustrial and academic applications is performed using Taq polymeraseand automated thermal cycling instrumentation.

In addition to Taq polymerase, other thermostable polymerases have foundsimilar application in PCR (Bej and Mahbubani, Id). Particularly usefulas substitutes for Taq polymerase in PCR are polymerases isolated fromthe thermophilic bacteria Thermus thermophilus (Tth polymerase),Thermococcus litoralis (Tli or VENTT™ polymerase), Pyrococcus furiosus(Pfu or DEEPVENT polymerase), Pyrococcuswoosii (Pwo polymerase) andother Pyrococcus species, Bacillus sterothermophilus (Bst polymerase),Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma acidophilum(Tac polymerase), Thermus flavus (Tfl/Tub polymerase), Thermus ruber(Tru polymerase), Thermus brockianus (DYNAZYME™ polymerase), Thermotoganeapolitana (Tne polymerase; See WO 96/10640), Thermotoga maritima (Tmapolymerase; See U.S. Pat. No. 5,374,553) and other species of theThermotoga genus (Tsp polymerase) and Methanobacteriumthermoautotrophicum (Mth polymerase). While each of these polymerases isuseful for particular applications (See Bej and Mahbubani, Id., p. 222),Taq polymerase is still by far the most commonly used polymerase in PCR.

Thermostable polymerases have also found application in DNA sequencingtechniques, particularly in automated methods of dideoxy sequencing suchas “cycle sequencing.” These approaches resemble PCR in most respectsexcept that, in place of dNTPs, automated DNA sequencing uses ddNTPswhich allow determination of the sequence of the template DNA asdescribed above. Use of higher denaturation temperatures in automatedsequencing also improves sequencing efficiency (i.e., fewermisincorporations occur) and allows the sequencing of templates that areGC-rich or contain significant secondary structure (such assupercoiling).

The use of thermolabile DNA polymerases such as E. coli or T7 DNApolymerases in these approaches, however, is subject to the samelimitations described above for their use in PCR. Accordingly, automatedmethods of DNA sequencing utilizing higher temperatures haveincreasingly employed thermostable DNA polymerases, the most commonlyused of which is, as for PCR, Taq polymerase.

Technical Limitations

The use of Taq and other thermostable polymerases in sequencing and PCRis not, however, without drawback. For example, the error rate for Taqpolymerase is substantially higher (i.e., the final product is of “lowerfidelity”) than that for most of the thermolabile DNA polymerases,including the Klenow fragment of E. coli DNA polymerase I (Tindall andKunkel, Id.), averaging about 10⁻⁴ misincorporations per base pair percycle. In addition, Taq polymerase is only useful for amplifyingrelatively short stretches of DNA (maximum length on the order of 5-6kilobases; Barnes, W. M., Proc. Natl. Acad. Sci. USA 91:2216-2220,1994), thus precluding its use in PCR amplification of large genes andwhole genomes as is necessary in many current applications.

These technical limitations are apparently related: it has beentheorized that the Taq polymerase PCR length limitation is due to thelow efficiency of elongation of the newly synthesized DNA strands at thesites of incorporation of mismatched bases in the parent strands(Barnes, Id). Further contributing to this difficulty is the absence inTaq polymerase of a 3′ to 5′ exonuclease activity, which in otherpolymerases acts in a “proofreading” capacity to correct thesemismatches and reduce the error rate (Bej and Mahbubani, Id). The 5′ to3′ exonuclease activity present in most thermostable DNA polymerases canalso degrade the 5′ ends of the oligonucleotide primers (also acomplication with the 3′ to 5′ exonuclease activity which can degradethe 3′ ends of the primers), yielding undesirable results due to anearly termination of the PCR process (See WO 92/06200; Barnes, Id).

In sequencing reactions, Taq polymerase is subject to a limitationshared by E. coli polymerase I and the Klenow fragment. These enzymeseach are “discriminatory,” meaning that they preferentially incorporatedNTPs over ddNTPs into newly synthesized DNA. Thus, to use Taqpolymerase in automated sequencing reactions, relatively highconcentrations of ddNTPs must be maintained in the reaction mixtures, tokinetically favor ddNTP incorporation by the enzyme. This need for highlevels of ddNTPs can be prohibitively expensive, particularly when largeDNA fragments are being sequenced.

As another technical limitation, the DNA polymerases have heretoforebeen maintained in highly concentrated stock solutions in storagebuffers containing glycerol, bovine serum albumin and/or otherstabilizing agents and stored at −20° C. or lower (See, e.g., WO92/06188; U.S. Pat. No. 5,436,149). The conventional understanding inthe field has been that the enzymes would rapidly lose activity at moredilute working concentrations (as do many bioactive proteins) and insolutions without glycerol or other stabilizing agents. Moreover, thesolutions of enzymes had to be mixed with dNTPs or ddNTPs, cofactors(such as Mg⁺⁺) and one or more detergents immediately prior to use inthe sequencing or PCR processes, as it was believed that premixture andstorage of these solutions would also deleteriously affect theirstability. In addition, dNTPs have traditionally been stored attemperatures below −20° C. and have also been thought to be unstable ifstored otherwise (Maniatis, T., et al., Molecular Cloning, A LaboratoryManual, CRC Press, 1992). Together, these limitations have made the useof compositions containing thermostable DNA polymerases in DNAsequencing and PCR more costly and time-consuming than would be desired.

Overcoming Technical Limitations

Several approaches have been undertaken to attempt to surmount thesetechnical difficulties. The results of DNA sequencing methodologies, forexample, have been improved by the use of mutant Taq enzymes such asATaq (WO 92/06188) lacking the 5′ to 3′ exonuclease activity. Improvedsequencing results have also been obtained by including in the reactionmixture an agent such as pyrophosphatase which breaks down thepyrophosphate that can be formed during dideoxy sequencing reactions(See U.S. Pat. No. 5,498,523). To overcome the discrimination betweenddNTPs and dNTPs, some investigators have used T7 DNA polymerase insequencing, as this enzyme is “nondiscriminatory,” meaning that itincorporates ddNTPs at approximately the same rate as dNTPs (See U.S.Pat. No. 4,795,699). Alternatively, mutants of DNA polymerase from avariety of organisms (e.g., E. coli) which are nondiscriminatory havealso been described; see copending U.S. patent application Ser. No.08/525,057 of Deb K. Chatterjee, filed Sep. 8, 1995, entitled “MutantDNA Polymerases and Use Thereof,” the disclosure of which is expresslyincorporated herein by reference. However, as described above, both E.coli and T7 DNA polymerases are thermolabile, so their use in automatedsequencing requires addition of fresh enzyme at the beginning of eachcycle.

More recently, mutations in The polymerase from Thermotoga neapolitanahave been described, which overcome these limitations (WO 96/10640). Oneof these mutations, in which a phenylalanine residue at amino acidposition number 730 in the wildtype protein (SEQ ID NO:1) is replacedwith a tyrosine residue, results in a mutant Tne polymerase (SEQ IDNO:2) which is both thermostable and nondiscriminatory. This mutant Tnepolymerase thus provides a solution to both the problems ofthermolability and ddNTP discrimination found in other enzymes used inautomated DNA sequencing. See also the co-pending U.S. patentapplication of A. John Hughes and Deb K. Chatterjee, entitled “ClonedDNA Polymerases from Thermotoga and Mutants Thereof,” filed on even dayherewith, which is incorporated by reference herein in its entirety.

In PCR applications, the low fidelity of Taq-produced PCR products hasbeen alleviated to some extent by the use of Pfu DNA polymerase whichcontains the proofreading 3′ to 5′ exonuclease activity lacking in Taqpolymerase (Lundberg, K. S., et al., Gene 108:1-6, 1991). Otherthermostable DNA polymerases, including Tli/VENT™ (Bej and Mahbubani,Id) and DEEPVENT™ (Flaman, J.-M., et al, Nucl. Acids Res.22(15):3259-3260, 1994) have also been shown to improve the fidelity ofPCR products.

As a means of overcoming this length limitation, mutant enzymes lackingthe 5′ to 3′ exonuclease activity have been prepared, includingN-terminal deletion mutants of Taq polymerase that are analogous to theKlenow fragment of E. coli DNA polymerase I. Several of these mutantenzymes, including Klentaq-1 and Klentaq-278 (Barnes, W. M., Gene112:29-35, 1992; U.S. Pat. No. 5,436,149), the Taq Stoffel fragment(Lawyer, F. C., et al., PCR Meth. Appl. 2:275-287, 1993), and mutants ofother thermostable DNA polymerases lacking the 5′ to 3′ exonucleaseactivity (e.g., those disclosed in WO 92/06200), have been shown toprovide increasingly stable PCR products and primers. However, as theseenzymes also lack the 3′ to 5′ proofreading activity, their use subjectsthe PCR process to the increased error rates described above. Thus, evenwith these mutant enzymes, high-fidelity PCR amplification of DNAfragments larger than 5-6 kilobases has proven exceedingly difficult.

By combining specific quantities of several enzymes, however,high-fidelity PCR amplification of large DNA sequences has beenachieved. For example, use of a combination of a high concentration of athermostable DNA polymerase lacking the 3′ to 5′ exonuclease activity(e.g., Klentaq278) and a low concentration of a thermostable DNApolymerase exhibiting the 3′ to 5′ exonuclease activity (e.g.,Pfu/DEEPVENT or Tli/VENT™) provides for amplification to highconcentrations of DNA sequences of at least 35 kilobases in length withsignificantly improved fidelity (Barnes, Id; U.S. Pat. No. 5,436,149).Similar results have been obtained with mixtures of Tth polymerase andlow levels of thermostable polymerases from Pyrococcus, Tli or Tma (U.S.Pat. No. 5,512,462). Apparently, the low level of 3′ to 5′ exonucleaseactivity is sufficient to remove any mismatched bases incorporated bythe majority polymerase, but is insufficient to significantly degradethe primers. While this approach has heretofore been applied to simpleDNA sequences such as those from bacteriophage λ, it may proveapplicable to larger and more complex sequences as well, including thoseof the genomes of bacteria, yeast, plants and animals.

Despite its widespread use, however, conventional PCR can producenon-specific amplification fragments which range from small primer-dimerproducts to target fragments of various yields and of heterogeneoussize. These non-specific products not only obscure PCR results, but canalso limit the sensitivity of PCR product detection and can alsointerfere with downstream processes such as DNA sequencing and cloningof PCR fragments. These artifact amplification products are often due tonon-specific annealing and extension of primers at low temperatures, andto the presence of a low level of polymerase activity in the reactionmixtures during setup and start of PCR (Li, H., et al., Proc. Natl.Acad. Sci. USA 87:4580 (1990); Frohman, M. A., et al., Proc. Natl. Acad.Sci. USA 85:8998 (1988); Chou, Q., et al., Nucl. Acids Res. 20:1717(1992)). Consequently, a number of physical manipulation methods havebeen developed to circumvent the non-specific priming during the PCRsetup and start of the reaction'. These manual methods are oftenreferred to as “Hot Start” and involve the addition of Taq DNApolymerase to preheated (typically to about 80° C.) PCR reactions (Chou,Q., et al., Nucl. Acids Res. 20:1717 (1992); D'Aquila, R. T., et al.Nucl. Acids Res. 19:3749 (1991). These methods, however, are oftencumbersome and are not used for routine or high throughput applications.

It has recently been shown that specific monoclonal antibodies tothermostable DNA polymerases can be used to improve specificity of PCRamplification (see U.S. Pat. No. 5,338,671, the disclosure of which isincorporated herein by reference in its entirety; Sharkey, D. J., etal., BioTechnology 12:506 (1994); Daiss, J. L. et al., J. Immunol. Meth.183:15 (1995)). These monoclonal antibodies prevent the polymerizationactivity of the enzyme and result in inactivity of Taq DNA polymeraseduring the PCR setup and start of reactions. However, during the initialdenaturation step of PCR, antibodies are denatured and active Taq DNApolymerase is released into the reaction. This approach provides aneffective and automatic method for control of non-specific PCR productsin all PCR reactions.

Despite these successes in overcoming the ddNTP discrimination, length,fidelity, and artifact limitations, however, compositions comprising thereagents necessary for DNA sequencing or PCR capable of extended storageabove freezing at working concentrations, without stabilizing agents,have not heretofore been reported. Thus, the time and economicconstraints to the use of solutions of thermostable enzymes in mostapplications have yet to be overcome.

BRIEF SUMMARY OF THE INVENTION

The present invention overcomes these temporal and economic limitationsof previously available reagent compositions used in nucleic acidamplification and sequencing methods. Specifically, the invention isdirected to compositions comprising mixtures of reagents at workingconcentrations suitable for use with or without dilution and maintainingactivity upon storage for an extended time, said mixtures consistingessentially of at least one thermostable enzyme and at least one buffersalt. The invention further provides such compositions for use innucleic acid amplification further comprising at least onedeoxynucleoside triphosphate, magnesium salts and at least one nonionicdetergent, wherein the thermostable enzyme is at least one thermostableDNA polymerase. In another embodiment, the invention provides suchcompositions for use in nucleic acid sequencing further comprising atleast one deoxynucleoside triphosphate, at least one dideoxynucleosidetriphosphate, magnesium salts and at least one nonionic detergent,wherein the thermostable enzyme is at least one thermostable DNApolymerase. More specifically, the invention is directed to suchcompositions wherein the thermostable DNA polymerase is Taq, Tne, Tma,Pfu, Pwo or Tth DNA polymerase, or mutants thereof, preferably atconcentrations of about 0.1-200 units per milliliter, about 0.1-50 unitsper milliliter, about 0.1-40 units per milliliter, about 0.1-36 unitsper milliliter, about 0.1-34 units per milliliter, about 0.1-32 unitsper milliliter, about 0.1-30 units per milliliter, or about 0.1-20 unitsper milliliter, and most preferably at concentrations of about 20 unitsper milliliter. The invention is also directed to such compositions thatfurther comprise VENT or DEEPVENT™ DNA polymerase, preferably atconcentrations of about 0.0002-200 units per milliliter, about 0.002-100units per milliliter, about 0.002-20 units per milliliter, about0.002-2.0 units per milliliter, about 0.002-1.6 units per milliliter,about 0.002-0.8 units per milliliter, about 0.002-0.4 units permilliliter, or about 0.002-0.2 units per milliliter, most preferably atconcentrations of about 0.40 units per milliliter.

In another embodiment, the invention is directed to such compositionswhich optionally further comprise at least one antibody whichspecifically binds to the one or more thermostable enzymes (such as theone or more DNA polymerases) in the compositions. The antibodies used inthis aspect of the invention may be polyclonal or monoclonal, and arepreferably monoclonal, and may include (but are not limited to) anti-DNApolymerase antibodies, particularly antibodies which bind specificallyto one or more thermostable DNA polymerases, such as anti-Taqantibodies, anti-Tne antibodies, anti-Tma antibodies, anti-Pfuantibodies, anti-Pwo antibodies, anti-Tth antibodies, and the like.Preferably, the antibodies are used in the compositions at an antibodyto polymerase concentration ratio of up to about 100:1, up to about50:1, up to about 25:1, up to about 20:1, up to about 15:1, up to about10:1, up to about 9:1, up to about 8:1, up to about 7.5:1, up to about7:1, up to about 6:1, up to about 5:1, up to about 4:1, up to about 3:1,up to about 2.5:1, up to about 2:1, or up to about 1:1. Most preferably,the antibodies are used in the compositions at an antibody to polymeraseconcentration ratio of about 1:1 to about 10:1, or about 1:1 to about5:1.

The invention is further directed to kits for DNA amplification orsequencing, said kits comprising a carrier means, such as a box, carton,tube or the like, having in close confinement therein one or morecontainer means, such as vials, tubes, ampules, bottles or the like,wherein a first container means contains a composition comprising amixture of reagents at working concentrations suitable for use withoutdilution and maintaining activity upon storage for extended time, saidmixture consisting essentially of at least one thermostable DNApolymerase, buffer salts, magnesium salts and at least one nonionicdetergent. In additional embodiments, the kits may optionally compriseone or more antibodies, in the first container means or in a separatecontainer means, which specifically bind to one or more of the DNApolymerases present in the compositions of the kits, such as thoseantibodies described above. The first container means may also contain amixture of dNTPs (for PCR applications) or ddNTPs (for sequencingapplications). Alternatively, the dNTPs or ddNTPs may be included in asecond container means also closely confined within the carrier means ofthe kit.

The invention is also directed to methods of amplifying or sequencing anucleic acid molecule, comprising contacting the nucleic acid moleculeto be amplified or sequenced, which is preferably larger than about 4-8kilobases in size, more preferably larger than about 5-7 kilobases insize and most preferably larger than about 7 kilobases in size, with thecompositions of the invention. The invention also provides nucleic acidmolecules amplified by these methods.

The present invention is also directed more generally to compositionscontaining thermostable proteins or enzymes useful in molecular biology.These compositions comprise mixtures of reagents at workingconcentrations suitable for use with or without dilution which maintainthe enzyme or protein activity upon storage for an extended time. Thecompositions of this aspect of the invention comprise at least onethermostable enzyme and at least one buffer salt. The thermostableenzymes in these compositions include, but are not limited to,polymerases, restriction enzymes, alkaline phosphatases, reversetranscriptases, ligases, nucleases, pyrophosphatases, DNAses, RNAses,exonucleases, RNAse inhibitors, kinases, topoisomerases,guanylyltransferases and glycosylases (e.g., uracil DNA glycosylase).

The invention is further directed to kits for conducting a procedureassociated with the thermostable enzymes or proteins (restrictionenzymes, phosphatases, etc.), the kits comprising a container means suchas a box, carton, tube and the like, having in close confinement thereinone or more container means, such as a vial, tube, ampule, bottle or thelike, wherein a first container means contains a composition comprisinga mixture of reagents at working concentrations suitable for use with orwithout dilution. The reagents in the first container means include atleast one thermostable enzyme or protein and at least one buffer salt.

The compositions of the invention have unexpectedly been found tomaintain enzyme activity for extended periods of time compared toconventional compositions. For example, the present compositionsmaintain enzyme activity for at least four weeks when stored at ambienttemperature (about 20°-25° C.), for at least one year at about 4° C. andfor at least two years at about −20° C.

These stable, ready-to-use reagent compositions are useful for nucleicacid amplification (including the Polymerase Chain Reaction or PCR) andsequencing (including dideoxy sequencing), or for any procedure usingthermostable DNA polymerases or other enzymes (restriction enzymes,phosphatases, kinases, etc.) in fields such as medical therapeutics anddiagnostics, forensics and agricultural science.

Other features, advantages and applications of the present inventionwill be apparent to those skilled in the art from the followingdescription of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of an agarose gel (visualized by ethidium bromidefluorescence under ultraviolet illumination) of PCR amplification of a1.3 kilobase human genomic DNA fragment from 10 nanograms of templateusing 15 different reagent compositions (corresponding to samples 10-24in Table 1) stored as indicated in Table 1, and a freshly made controlsample. Lane marked “M” contains markers indicating amount of DNAloaded; bands correspond to (from top to bottom) 100 nanograms, 60nanograms, 40 nanograms and 20 nanograms of DNA mass markers.

FIG. 2 is a photograph of an agarose gel (visualized by ethidium bromidefluorescence under ultraviolet illumination) of PCR amplification of a4.1 kilobase human genomic DNA fragment using the amounts indicated (innanograms) of template for each amplification reaction. The samples onthe upper portion of the gel were amplified with a nucleic acidamplification composition stored for 10 weeks at 4° C., while those onthe lower portion of the gel were amplified with a composition storedfor 10 weeks at ambient temperature (about 20-25° C.). Leftmost lanes ineach portion are markers as indicated in FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Throughout this disclosure, various terms that are generally understoodby those of routine skill in the art are used. Certain terms as usedherein, however, have specific meanings for the purposes of the presentinvention. The term “dNTP” (plural “dNTPs”) generically refers to thedeoxynucleoside triphosphates (e.g., dATP, dCTP, dGTP, dTTP, dUTP, dITP,7-deaza-dGTP, adATP, adTTP, adGTP and adCTP), and the term “ddNTP”(plural “ddNTPs”) to their dideoxy counterparts, that are incorporatedby polymerase enzymes into newly synthesized nucleic acids. The term“unit” as used herein refers to the activity of an enzyme. Whenreferring to a thermostable DNA polymerase, one unit of activity is theamount of enzyme that will incorporate 10 nanomoles of dNTPs intoacid-insoluble material (i.e., DNA or RNA) in 30 minutes under standardprimed DNA synthesis conditions. “Working concentration” is used hereinto mean the concentration of a reagent that is at or near the optimalconcentration used in a solution to perform a particular function (suchas amplification, sequencing or digestion of nucleic acids). The term“detergent” as used herein refers to a nonionic surfactant such asTRITON X-100®, Nonidet P-40 (NP-40), Tween 20 or Brij 35. The terms“stable” and “stability” as used herein generally mean the retention byan enzyme of at least 70%, preferably at least 80%, and most preferablyat least 90%, of the original enzymatic activity (in units) after theenzyme or composition containing the enzyme has been stored for at leastfour weeks at a temperature of about 20-25° C., at least one year at atemperature of about 4° C. or at least 2 years at a temperature of −20°C.

Overview

The present invention provides, in a first preferred embodiment,compositions comprising mixtures of at least one thermostable enzyme(e.g., a thermostable DNA polymerase, restriction enzyme, etc.), atleast one buffer salt, and other reagents necessary for carrying out theprocedure associated with the enzyme(s) (e.g., deoxynucleosidetriphosphates (dNTPs) for amplification of nucleic acids, dNTPs anddideoxynucleoside triphosphates (ddNTPs) for sequencing of nucleicacids, etc.). In additional preferred embodiments, the inventionprovides such compositions which may further comprise one or moreantibodies which specifically bind to the one or more thermostableenzymes (such as the one or more DNA polymerases) in the compositions.The compositions of the invention contain no stabilizing compounds(e.g., glycerol, serum albumin or gelatin) that have been traditionallyincluded in stock reagent solutions, and exhibit increased stability(measured as maintenance of enzyme activity) even upon storage attemperatures above freezing. Furthermore, the invention provides thesereagent compositions in ready-to-use concentrations, obviating thetime-consuming dilution and pre-mixing steps necessary with previouslyavailable solutions. Unexpectedly, even at these diluted concentrationsthe reagent compositions are stable for extended periods of time attemperatures ranging from ambient (about 20-25° C.) to about −70° C.

In additional preferred embodiments, the present invention providesthese ready-to-use compositions in the form of kits that are suitablefor immediate use to carry out the procedure associated with theenzyme(s) (e.g., nucleic acid amplification or sequencing in the case ofDNA polymerases). These kits are also stable for extended periods oftime at temperatures ranging from ambient (about 20-25° C.) to −70° C.

In additional preferred embodiments, the invention provides ready-to-usecompositions for PCR amplification. The ready-to-use reagents willcontain all necessary components for PCR amplification such as one ormore DNA polymerase(s), one or more deoxynucleoside triphosphates(dNTPs) and buffers, and optionally one or more other componentscontributing to efficient amplification of nucleic acid templates byautomatic “hot start.” Automatic Hot Start PCR can be accomplished byreaction of specific antibodies, e.g., monoclonal antibodies, that bindto and inactivate one or more DNA polymerases, such as thermostable DNApolymerases (e.g., Taq DNA polymerase), that are present in theready-to-use compositions of the invention. In additional embodiments,the invention provides formulation of ready-to-use PCR reagents whichcontain one or more thermostable DNA polymerases (e.g., Taq DNApolymerase), one or more dNTPs, one or more buffers, and one or moreantibodies that bind to a DNA polymerase.

Sources of Reagents

The compositions of the present invention may be formed by mixing thecomponent reagents at the concentrations described below. The componentsfor making the ready-to-use compositions can be obtained from, forexample, Life Technologies, Inc. (Rockville, Md.).

Thermostable Enzymes

The thermostable enzymes (e.g., DNA polymerases, restriction enzymes,phosphatases, etc.) used in the present invention may be isolated fromnatural or recombinant sources, by techniques that are well-known in theart (See Bej and Mahbubani, Id.; WO 92/06200; WO 96/10640), from avariety of thermophilic bacteria that are available commercially (forexample, from American Type Culture Collection, Rockville, Md.) or maybe obtained by recombinant DNA techniques (WO 96/10640). Suitable foruse as sources of thermostable enzymes or the genes thereof forexpression in recombinant systems are the thermophilic bacteria Thermusthermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcuswoosii and other species of the Pyrococcus genus, Bacillussterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum,Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoganeapolitana, Thermotoga maritima and other species of the Thermotogagenus, and Methanobacterium thermoautotrophicum, and mutants thereof. Itis to be understood, however, that thermostable enzymes from otherorganisms may also be used in the present invention without departingfrom the scope or preferred embodiments thereof. As an alternative toisolation, thermostable enzymes (e.g., DNA polymerases) are availablecommercially from, for example, Life Technologies, Inc. (Rockville,Md.), New England Biolabs (Beverly, Mass.), Finnzymes Oy (Espoo,Finland) and Perkin Elmer Cetus (Norwalk, Conn.). Once obtained, thepurified enzymes may be placed into solution at working concentrationsand stored according to the methods of the present invention.

dNTPs

The dNTP components of the present compositions serve as the “buildingblocks” for newly synthesized nucleic acids, being incorporated thereinby the action of the polymerases. These dNTPs deoxyadenosinetriphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxyguanosinetriphosphate (dGTP), deoxythymidine triphosphate (dTTP), and for someapplications deoxyuridine triphosphate (dUTP) and deoxyinosinetriphosphate (dITP), α-thio-dATP and 7-deaza-dGTP—are availablecommercially from sources including Life Technologies, Inc. (Rockville,Md.), New England Biolabs (Beverly, Mass.) and Sigma Chemical Company(Saint Louis, Mo.). The dNTPs may be unlabeled, or they may bedetectably labeled by coupling them by methods known in the art withradioisotopes (e.g., ³H, ¹⁴C, ³²P or ³⁵S), vitamins (e.g., biotin),fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, orphycoerythrin) or other detection agents. Labeled dNTPs may also beobtained commercially, for example from Life Technologies, Inc.(Rockville, Md.) or Sigma Chemical Company (Saint Louis, Mo.). Onceobtained, the dNTPs may be placed into solution at workingconcentrations and stored according to the methods of the presentinvention.

ddNTPs

The ddNTP components of the present compositions serve as the“terminating agents” in the dideoxy nucleic acid sequencingmethodologies, being incorporated into newly synthesized nucleic acidsby the action of the polymerases. These ddNTPs—dideoxyadenosinetriphosphate (ddATP), dideoxycytosine triphosphate (ddCTP),dideoxyguanosine triphosphate (ddGTP), dideoxythymidinetriphosphate(ddTTP), and for some applications dideoxyuridine triphosphate (ddUTP)and dideoxyinosine triphosphate (ddITP)—are available commercially fromsources including Life Technologies, Inc. (Rockville, Md.), New EnglandBiolabs (Beverly, Mass.) and Sigma Chemical Company (Saint Louis, Mo.).The ddNTPs may be unlabeled, or they may be detectably labeled bycoupling them by methods known in the art with radioisotopes (e.g., ³H,¹⁴C, ³²P or ³⁵S), vitamins (e.g., biotin), fluorescent moieties (e.g.,fluorescein, rhodamine, Texas Red, or phycoerythrin) or other detectionagents. Labeled ddNTPs may also be obtained commercially, for examplefrom Life Technologies, Inc. (Rockville, Md.) or Sigma Chemical Company(Saint Louis, Mo.). Once obtained, the ddNTPs may be placed intosolution at working concentrations and stored according to the methodsof the present invention.

Buffers/Salts

All buffers and cofactor salts comprising the compositions of thepresent invention, and concentrated stock solutions thereof areavailable from a variety of commercial sources including LifeTechnologies, Inc. (Rockville, Md.) and Sigma Chemical Company (SaintLouis, Mo.). Particularly preferred buffers for use in forming thepresent compositions are the sulfate, hydrochloride, phosphate or freeacid forms of tris-(hydroxymethyl)aminomethane (TRIS®), althoughalternative buffers of the same approximate ionic strength and pKa asTRIS® may be used with equivalent results. In addition to the buffersalts, cofactor salts such as those of potassium (preferably potassiumchloride) and magnesium (preferably magnesium chloride or sulfate) areincluded in the compositions. Once obtained, the buffers and cofactorsalts may be placed into solution at working concentrations and storedaccording to the methods of the present invention.

Detergents

At least one detergent may be included as a component of the presentcompositions, to provide for both increased stability and activity ofthe component enzymes. Nonionic detergents are preferred, to maintain abalanced ionic strength and prevent chelation of cofactors andaggregation or inactivation of proteins. Particularly preferred asdetergents are TRITON X-100®, Brij 35, Tween 20 and Nonidet P-40(NP-40), although other nonionic surfactants and mixtures thereof mayalso be used in the present compositions. These detergents are availablecommercially from sources such as Sigma Chemical Company (Saint Louis,Mo.), usually as concentrated aqueous solutions or in powder form. Onceobtained, the detergents may be placed into solution at workingconcentrations and stored according to the methods of the presentinvention.

Antibodies

In additional embodiments of the invention, the compositions mayoptionally comprise one or more antibodies which specifically bind tothe one or more thermostable enzymes, such as the one or more DNApolymerases, present in the compositions of the invention. According tothis aspect of the invention, the one or more antibodies willspecifically bind to the one or more thermostable enzymes (such as theone or more DNA polymerases) at temperatures below about 45° C.; as aresult of this binding, the enzymatic activity of the enzyme will becompletely or substantially completely inhibited. However, once thecomposition or reaction mixture containing the composition is raised toa temperature above about 60-65° C. (e.g., the temperatures at whichstandard PCR methods are conducted), the antibody is denatured and theactivity of the enzyme is restored. Thus, such compositions will haveutility in such applications as “Hot Start” PCR amplification protocols.Antibodies for use in this aspect of the invention include polyclonalantibodies, monoclonal antibodies, and enzyme-binding fragments (such asF(ab′) or F(ab′)₂ fragments) thereof.

According to the invention, any antibody or fragment thereof whichspecifically binds to one or more of the thermostable enzymes in thepresent compositions, such as the DNA polymerases, may be used,including but not limited to anti-Taq antibodies, anti-Tne antibodies,anti-Tma antibodies, anti-Pfu antibodies, anti-Pwo antibodies, anti-Tthantibodies, and the like. These and other antibodies suitable for use inthis aspect of the invention may be obtained commercially, e.g., fromLife Technologies, Inc. (Rockville, Md.). Alternatively, antibodies maybe produced in animals by routine methods of production of polyclonalantibodies (see, e.g., Harlow, E., and Lane, D., Antibodies: ALaboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1988); Kaufman, P. B., et al, In: Handbook ofMolecular and Cellular Methods in Biology and Medicine, Boca Raton,Fla.: CRC Press, pp. 468-469 (1995) or monoclonal antibodies (see, e.g.,Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., In: Monoclonal Antibodies and T-Cell Hybridomas, New York:Elsevier, pp. 563-681 (1981); Kaufman, P. B., et al., In: Handbook ofMolecular and Cellular Methods in Biology and Medicine, Boca Raton,Fla.: CRC Press, pp. 444-467 (1995)), using the correspondingthermostable enzyme (such as the corresponding DNA polymerase) as animmunogen.

Formulating the Reagent Compositions

Once the reagent components have been obtained, they are mixed atworking concentrations to form a solution suitable for immediate usewith or without dilution or addition of further reagents. The water usedin the formulations of the present invention is preferably distilled,deionized and sterile filtered (through a 0.1-0.2 micrometer filter),and is free of contamination by DNase and RNase enzymes. Such water isavailable commercially, for example from Sigma Chemical Company (SaintLouis, Mo.), or may be made as needed according to methods well known tothose skilled in the art.

Although the components of the present compositions may be admixed inany sequence, it is often preferable to first dissolve the buffer(s) andcofactor salts in water and to adjust the pH of the solution prior toaddition of the remaining components. In this way, the pH-sensitivecomponents (particularly the enzymes, ddNTPs and dNTPs) will be lesssubject to acid- or alkaline-hydrolysis during formulation.

To formulate the buffered salts solution, a buffer salt which ispreferably a salt of tris(hydroxymethyl)aminomethane (TRIS®), and mostpreferably the hydrochloride salt thereof, is combined with a sufficientquantity of water to yield a solution having a TRIS® concentration of5-150 millimolar, preferably 10-60 millimolar, and most preferably about20-60 millimolar. To this solution, a salt of magnesium (preferablyeither the chloride or sulfate salt thereof) may be added to provide aworking concentration thereof of 1-10 millimolar, preferably 1.5-5millimolar, and most preferably about 1.5-2 millimolar. A salt ofpotassium (most preferably potassium chloride) may also be added to thesolution, at a working concentration of 10-100 millimolar and mostpreferably about 50 millimolar. An ammonium salt, for example ammoniumsulfate, may also be added to the mixture, at a working concentration of2-50 millimolar, preferably 10-30 millimolar and most preferably 18millimolar. Combinations of ammonium sulfate and potassium chloride (orother salts) may also be used in formulating the compositions of thepresent invention. A small amount of a salt ofethylenediaminetetraacetate (EDTA) may also be added (preferably about0.1 millimolar), although inclusion of EDTA does not appear to beessential to the function or stability of the compositions of thepresent invention. After addition of all buffers and salts, thisbuffered salt solution is mixed well until all salts are dissolved, andthe pH is adjusted using methods known in the art to a pH value of 7.4to 9.2, preferably 8.0 to 9.0, and most preferably about 8.3 forcompositions to be used in amplification or sequencing of nucleotidefragments up to about 5-6 kilobases in size (hereinafter referred to as“standard compositons”), and about 8.9 for compositions to be used foramplification or sequencing of nucleotide fragments larger than about5-6 kilobases in size (hereinafter referred to as “large sequencecompositions”).

To the buffered salt solution, the remaining components of the presentcomposition are added. It is well known in the field that the additionof one or more detergents to an aqueous buffer will aid in thesubsequent solubilization of added proteins. Accordingly, at least onenonionic detergent such as TRITON X-100® (preferably at a workingconcentration of 0.1-1%), Brij 35 (preferably at a concentration of0.01-1% and most preferably of about 0.1%) or Nonidet P-40 (NP-40,preferably as an admixture with a concentration of 0.004-1%, and mostpreferably in admixture with Tween 20 at a working concentration of 0.1%for standard compositions and 0.02% for large sequence compositions) maybe added to the buffer solution. This detergent is preferably addedprior to the introduction of the remaining components into the solution,although the detergent may equivalently be added at any step offormulation. Following formulation, the buffered salt solutions may befiltered through a low protein-binding filter unit that is availablecommercially (for example from Millipore Corporation, Bedford, Mass.)and stored until use.

The remaining components are then added to the solution to formulate thecompositions of the present invention. At least one thermostable enzyme(e.g., DNA polymerase) is added and the solution is gently mixed (tominimize protein denaturation). For standard DNA amplification(including via PCR) or sequencing of DNA segments up to about 5-6kilobases in length, any thermostable DNA polymerase (hereinafter the“primary polymerase”) may be used in the standard compositions, althoughTaq, Tne, Tma, VENT™, DEEPVENT™, Pfu or Pwo polymerases are preferableat a working concentration in the solution of about 0.1-200 units permilliliter, about 0.1-50 units per milliliter, about 0.1-40 units permilliliter, about 0.1-36 units per milliliter, about 0.1-34 units permilliliter, about 0.1-32 units per milliliter, about 0.1-30 units permilliliter, or about 0.1-20 units per milliliter, and most preferably ata working concentration of about 20 units per milliliter. Foramplification of DNA segments larger than 5-6 kilobases in length, largesequence compositions should be formulated by adding to the standardcompositions a low concentration of one or more additional thermostableDNA polymerases (hereinafter the “secondary polymerase”) containing a3′-5′ exonuclease activity. Particularly suited for this application areVENT™, Pfu, Pwo or Tne, and most preferably DEEPVENT™, DNA polymerases.The additional polymerase(s) should be added to the solution insufficient quantity to give a final working concentration of about0.0002-200 units per milliliter, about 0.002-100 units per milliliter,about 0.002-20 units per milliliter, about 0.002-2.0 units permilliliter, about 0.002-1.6 units per milliliter, about 0.002-0.8 unitsper milliliter, about 0.002-0.4 units per milliliter, or about 0.002-0.2units per milliliter, most preferably at concentrations of about 0.40units per milliliter.

It has heretofore been thought that the activity ratios of the primaryto secondary polymerases should be maintained at 4:1-2000:1 for largesequence amplification (see U.S. Pat. No. 5,436,149). It has now beendiscovered, however, that in the compositions of the present inventionthat activity ratios of the primary to secondary polymerases of 1:1,1:2, 1:4, 1:5, 1:8, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000 and1:2000 may be suitable for amplification of large nucleotide sequences.

For nucleic acid sequencing, the reagent compositions may be used asformulated above. For nucleic acid sequencing by the dideoxy method (SeeU.S. Pat. Nos. 4,962,020, 5,173,411 and 5,498,523), however, preferablythe mutant Tne DNA polymerase shown in SEQ ID NO:2 is added to thereagent compositions. Tne polymerase is added to the solution to give aworking concentration of about 0.1-10,000 units per milliliter, about0.1-5000 units per milliliter, about 0.1-2500 units per milliliter,about 0.1-2000 units per milliliter, about 0.1-1500 units permilliliter, about 0.1-1000 units per milliliter, about 0.1-500 units permilliliter, about 0.1-300 units per milliliter, about 0.1-200 units permilliliter, about 0.1-100 units per milliliter, or about 0.1-50 unitsper milliliter, and most preferably of about 300 units per milliliter.

For dideoxy sequencing, a solution of each ddNTP is also prepared. Thebase of each solution contains dATP, dCTP, dTTP, 7-deaza-GTP and/orother dNTPs, each at a working concentration of about 10-1000micromolar, about 10-500 micromolar, about 10-250 micromolar, or about10-100 micromolar, most preferably at a concentration of about 100micromolar, in a solution of buffer and chelating salts, for exampleTRIS®-HCl most preferably at a working concentration of about 10millimolar (pH about 7.5) and disodium-EDTA most preferably at aconcentration of about 0.1 millimolar. To this base, one of the ddNTPsis added to make each of four solutions. Preferably, the sodium orlithium salt of ddATP, ddCTP, ddGTP or ddTTP is added to the solution togive a working concentration of the ddNTP of about 0.5-10 micromolar,about 0.5-8 micromolar, about 0.5-5 micromolar, about 0.5-3 micromolar,about 0.5-2.5 micromolar, or about 0.5-2 micromolar, and most preferablyabout 2 micromolar. For cycle sequencing applications, the pH of theddNTP solutions will preferably be about 9.0, and the concentrations ofddNTPs may be lower, preferably about 0.05 to 1.0 micromolar or about0.05 to 0.8 micromolar, and most preferably about 0.08 to 0.8micromolar. For some applications, it may be desirable to alsoincorporate or substitute ddITP, ddUTP, and/or α-thio-dATP into thecompositions at approximately the same working concentrations. Thus,four solutions are prepared, each containing one of the four ddNTPs,which are combined with the polymerase compositions of the presentinvention to carry out the four separate reactions used in dideoxysequencing. Alternatively, for single-solution sequencing as disclosedin U.S. Pat. Nos. 4,962,020 and 5,173,411, the four ddNTPs may becombined into a single solution which is added to the polymerasecompositions of the present invention to perform the sequencingreaction.

For nucleic acid amplification, including PCR, dNTP salts are added tothe reagent compositions. Preferably, the sodium or lithium salts ofdATP, dCTP, dGTP and dTTP are added to the solution to give a workingconcentration of each dNTP of 10-1000 micromolar, preferably 200-300micromolar, and most preferably about 200 micromolar. For someapplications, it may be desirable to also incorporate or substitute dITPor dUTP into the compositions at the same working concentrations.

In certain embodiments as noted above, one or more antibodies thatspecifically bind to the one or more thermostable enzymes in thecompositions, such as the one or more DNA polymerases, may optionally beadded to the compositions. Preferably, the antibodies are used in thesecompositions at an antibody to polymerase concentration ratio of up toabout 100:1, up to about 50:1, up to about 25:1, up to about 20:1, up toabout 15:1, up to about 10:1, up to about 9:1, up to about 8:1, up toabout 7.5:1, up to about 7:1, up to about 6:1, up to about 5:1, up toabout 4:1, up to about 3:1, up to about 2.5:1, up to about 2:1, or up toabout 1:1. Most preferably, the antibodies are used in the compositionsat an antibody to polymerase concentration ratio of about 1:1 to about10:1, or about 1:1 to about 5:1.

To reduce component denaturation, storage of the reagent compositions ispreferably in conditions of diminished light, e.g., in amber orotherwise opaque containers or in storage areas with controlled lowlighting. The ready-to-use reagent compositions of the present inventionare unexpectedly stable at ambient temperature (about 20°-25° C.) forabout 4-10 weeks, are stable for at least one year upon storage at 4°C., and for at least two years upon storage at −20° C. Surprisingly,storage of the compositions at temperatures below freezing (e.g., −20°C. to −70° C.), as is traditional with stock solutions of bioactivecomponents, is not necessary to maintain the stability of thecompositions of the present invention.

In other preferred embodiments, the compositions of the presentinvention may be assembled into kits for use in nucleic acidamplification or sequencing. Sequencing kits according to the presentinvention comprise a carrier means, such as a box, carton, tube or thelike, having in close confinement therein one or more container means,such as vials, tubes, ampules, bottles and the like, wherein a firstcontainer means contains a stable composition comprising a mixture ofreagents, at working concentrations, which are at least one thermostableDNA polymerase, at least one buffer salt, at least one deoxynucleosidetriphosphate, at least one dideoxynucleoside triphosphate, andoptionally at least one antibody which specifically binds to at leastone thermostable DNA polymerase present in the compositions. Thesequencing kits may further comprise additional reagents and compoundsnecessary for carrying out standard nucleic sequencing protocols, suchas pyrophosphatase, agarose or polyacrylamide media for formulatingsequencing gels, and other components necessary for detection ofsequenced nucleic acids (See U.S. Pat. Nos. 4,962,020 and 5,498,523,which are directed to methods of DNA sequencing).

Similarly, amplification kits according to the present inventioncomprise carrier means, such as a box, carton, tube or the like, havingin close confinement therein one or more container means, such as vials,tubes, ampules, bottles and the like, wherein a first container meanscontains a stable composition comprising a mixture of reagents, atworking concentrations, which are at least one thermostable DNApolymerase, at least one buffer salt, at least one deoxynucleosidetriphosphate, and optionally at least one antibody which bindsspecifically to at least one thermostable DNA polymerase present in thecomposition. The amplification kits encompassed by this aspect of thepresent invention may further comprise additional reagents and compoundsnecessary for carrying out standard nucleic amplification protocols (SeeU.S. Pat. Nos. 4,683,195 and 4,683,202, which are directed to methods ofDNA amplification by PCR).

Use of the Reagent Compositions

The compositions and kits embodied in the present invention will havegeneral utility in any application utilizing nucleic acid sequencing oramplification methodologies. Amplification methods in which the presentcompositions may be used include PCR (U.S. Pat. Nos. 4,683,195 and4,683,202), Strand Displacement Amplification (SDA; U.S. Pat. No.5,455,166; EP 0 684 315), and Nucleic Acid Sequence-Based Amplification(NASBA; U.S. Pat. No. 5,409,818; EP 0 329 822). Nucleic acid sequencingtechniques which may employ the present compositions include dideoxysequencing methods such as those disclosed in U.S. Pat. Nos. 4,962,022and 5,498,523, as well as more complex PCR-based nucleic acidfingerprinting techniques such as Random Amplified Polymorphic DNA(RAPD) analysis (Williams, J. G. K., et al., Nucl. Acids Res.18(22):6531-6535, 1990), Arbitrarily Primed PCR (AP-PCR; Welsh, J., andMcClelland, M., Nucl. Acids Res. 18(24):7213-7218, 1990), DNAAmplification Fingerprinting (DAF; Caetano-Anolles et al.,Bio/Technology 9:553-557, 1991), microsatellite PCR or DirectedAmplification of Minisatellite-region DNA (DAVID; Heath, D. D., et al.,Nucl. Acids Res. 21(24): 5782-5785, 1993), and Amplification FragmentLength Polymorphism (AFLP) analysis (EP 0 534 858; Vos, P., et al.,Nucl. Acids Res. 23(21):4407-4414, 1995; Lin, J. J., and Kuo, J., FOCUS17(2):66-70, 1995). In particular, the compositions and kits of thepresent invention will be useful in the fields of medical therapeuticsand diagnostics, forensics, and agricultural and other biologicalsciences, in any procedure utilizing thermostable DNA polymerases.Furthermore, the methods by which the compositions of the presentinvention are formulated may be extendable to all thermostable enzymesor mixtures thereof, and may allow the formulation of ready-to-usecompositions of a variety of bioactive enzymes or other proteins thatdemonstrate increased stability upon extended storage at temperaturesabove freezing.

The compositions and kits of the invention are particularly useful inmethods for amplifying and sequencing nucleic acid molecules. Nucleicacid amplification methods according to this aspect of the inventioncomprise contacting a nucleic acid molecule to be amplified with one ormore of the compositions of the invention, thus providing a populationof amplified copies of the nucleic acid molecule. Nucleic acidsequencing methods according to this aspect of the invention comprisecontacting the nucleic acid molecule to be sequenced with one or more ofthe compositions of the invention. According to these methods,amplification and sequencing of the nucleic acid molecule may beaccomplished by any of the above-described amplification and sequencingtechniques, most preferably by PCR. The present amplification andsequencing methods are particularly useful for amplification andsequencing of large nucleic acid molecules (e.g., by “long PCR”),preferably nucleic acid molecules that are larger than about 4-8kilobases in size, more preferably larger than about 5-7 kilobases insize, and most preferably nucleic acid molecules that are larger thanabout 7 kilobases in size.

It will be readily apparent to those of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

Example 1 Formulation of Standard Compositions

As an initial step in formulating stable, ready-to-use reagentcompositions for nucleic acid amplification and sequencing, componentswere mixed in varying amounts as shown in Table 1 to provide 24different formulations. The pH on all formulations was adjusted to about8.3. After filtration through a low protein-binding filter, allcompositions were formulated with 20 units/ml of Taq polymerase and weresuitable for use as standard compositions for amplification orsequencing of nucleic acid fragments up to about 5-6 kilobases in size.

TABLE 1 FORMULATIONS OF STANDARD COMPOSITIONS Formulation Number 1 2 3 45 6 7 8 Tris-HCl,  20 mM  20 mM  20 mM  20 mM  20 mM  20 mM  20 mM  20mM pH 8.4 KCl  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM(NH₄)₂SO₄  0  0  0  0  0  0  0  0 dNTP 200 μM 200 μM 200 μM 200 μM 200μM Na 200 μM Na 200 μM Li 200 μM Li MgCl₂  1.5 mM  1.5 mM  1.5 mM  1.5mM  1.5 mM  1.5 mM  1.5 mM  1.5 mM Detergents 0.004% 0.1% 0.1% 0.1% 0.1%0.004% 0.1% 0.004% Tween20/NP40 Tween20/NP40 Tween20/NP40 Tween20/NP40Tween20/NP40 Tween20/NP40 Tween20/NP40 Tween20/NP40 Taq, U/ml 20 20 2020 20 20 20 20 Formulation Number 9 10 11 12 13 14 15 16 17 Tris-HCl, 20 mM  20 mM  20 mM  20 mM  20 mM  20 mM  20 mM  20 mM  20 mM pH 8.4KCl  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM(NH₄)₂SO₄  0  0  0  0  0  0  0  0  0 dNTP 200 μM 200 μM 200 μM 200 μM200 μM 200 μM 200 μM 200 μM 200 μM MgCl₂  1.5 mM  3 mM  3 mM  3 mM  3 mM 3 mM  3 mM  3 mM  3 mM Detergents 0.1% 0.1% 0.1% Brij35 0.1% 0.01%0.01% 0.01% 1% 1% Brij35 Tween20/NP40 Tween20/NP40 TritonX-100Tween20/NP40 Brij35 TritonX-100 Tween20/NP40 Taq, U/ml 20 20 20 20 20 2020 20 20 Formulation Number 18 19 20 21 22 23 24 Tris-HCl, pH 8.4  20 mM 20 mM  20 mM  20 mM  20 mM  50 mM (pH 9.0)  20 mM (pH 8.8) KCl  50 mM 50 mM  50 mM  50 mM  50 mM  0  10 mM (NH₄)₂SO₄  0  0  0  0  0  20 mM 10 mM dNTP 200 μM 200 μM 200 μM 200 μM 200 μM 200 μM 200 μM MgCl₂  3 mM 3 mM  3 mM  3 mM  3 mM  1.5 mM  2 mM Detergents 1% TritonX-100 none0.1% 0.1% 0.1% 0.1% 0.1% Tween20/NP40 Tween20/NP40 Tween20/NP40Tween20/NP40 TritonX100 Taq, U/ml 20 20 20 20 20 20 20

Example 2 Stability of Standard Compositions

To examine the stability of the standard compositions formulated inExample 1, samples of each formulation were aliquoted and stored, underdiminshed light, at ambient temperature (about 20°-25° C.), 4° C., −20°C. and −70° C. Samples of each formulation from each temperature weretaken daily for the first week, and weekly thereafter, and used instability assays. These stability assays were performed by amplifying,via standard PCR, suboptimal amounts of human genomic DNA as a templateusing a template titration (if few samples were to be compared in a timepoint) or a fixed amount of template (if a larger number of samples wereto be compared). To the desired amount of template DNA in a givenformulation, 10 picomoles of primer was added, and the reaction mixtureswere subjected to 35 cycles of PCR of 30 seconds at 94° C., 30 secondsat 55° C. and 1 minute/kilobase at 72° C. A portion of each reaction wasthen subjected to agarose gel electrophoresis and visualized by ethidiumbromide fluorescence under ultraviolet illumination.

The results of these assays indicated that certain of the formulationsdemonstrated enhanced stability upon storage. As shown in FIG. 1, afterabout three months of storage at 4° C., formulations 15 and 19 hadcompletely lost enzymatic activity, as evidenced by an absence of bandsin the lanes corresponding to these samples. At this same time point,however, formulations 10-14, 16-18 and 20-24 demonstrated about the samelevels of enzymatic activity as a freshly made (“control”) formulation.

Storage temperature was also found to have a significant effect upon thestability of the formulations, even within a given formulation. As shownin FIG. 2, when samples of formulation 4 were examined after about 15weeks of storage at either 4° C. or about 20-25° C., the samples storedat 4° C. retained full enzymatic activity when compared to a controlsample. Those stored at about 20-25° C., however, had lost someactivity, as indicated by the lower yields of the target fragmentobtained at all template concentrations.

The results for all of the formulations at the various storagetemperatures are summarized in Table 2.

TABLE 2 STABILITY OF STANDARD COMPOSITIONS Storage Formulation Number¹Temperature, ° C. 1 2 3 4 5 6 7 8 20-25 <12 weeks <12 weeks <15 weeks<12 weeks nd² nd nd nd  4 >26 weeks >26 weeks >56 weeks >27 weeks >8weeks >8 weeks >8 weeks >8 weeks −20 >13 weeks >13 weeks nd >15 weeks ndnd nd nd −70 nd nd nd >15 weeks nd nd nd nd Storage Formulation Number¹Temperature, ° C. 9 10 11 12 13 14 15 16 17 20-25 nd <10 weeks  <6 weeks <6 weeks <12 weeks <10 weeks <7 days <12 weeks  <6 weeks  4 >40weeks >26 weeks >26 weeks >26 weeks >26 weeks >26 weeks <1 day >26weeks >26 weeks −20 >10 weeks >26 weeks >26 weeks >26 weeks >26weeks >26 weeks <7 days >26 weeks >26 weeks −70 nd nd nd nd nd nd nd ndnd Storage Formulation Number¹ Temperature, ° C. 18 19 20 21 22 23 2420-25 <6 weeks 0 days <10 weeks <10 weeks <12 weeks  <6 weeks <12 weeks 4 >26 weeks 0 days >26 weeks >26 weeks >13 weeks >26 weeks >26 weeks−20 >26 weeks 0 days >26 weeks >26 weeks >26 weeks >26 weeks >26 weeks−70 nd nd nd nd nd nd nd ¹Formulation numbers correspond to those inTable 1; ²nd = not done.These results indicate that several of the compositions unexpectedlymaintained enzymatic activity for 6-12 weeks upon storage at 20-25° C.,and for over one year at 4° C. Formulation 19, however, had completelylost activity within 24 hours of formulation. Formulation 15 alsoexhibited a rapid loss of activity.

For further analysis of the stability of these ready-to-usecompositions, several formulations were stored at 20-25° C. or at 4° C.for up to six months, with samples taken monthly for stability assaysperformed by a determination of polymerase unit activity. The results ofthese assays are summarized in Tables 3 and 4.

TABLE 3 STABILITY OF STANDARD COMPOSITIONS (PERCENTAGE OF ENZYMEACTIVITY REMAINING) UPON STORAGE AT 20-25° C. Formulation No.¹ 1 Month 2Months 3 Months 4 Months 10 89 65  nd² nd 11 106 3 nd nd 12 106 91 nd nd13 93 72 nd nd 14 91 76 nd nd 15 78 63 nd nd 16 94 85 nd nd 17 89 90 ndnd 18 90 84 nd nd 19 0 0 nd nd 20 88 81 72 77 21 nd 103 nd nd 22 97 84nd nd 23 83 77 nd nd 24 81 106 nd nd ¹Formulation numbers correspond tothose in Table 1. ²nd = not done

TABLE 4 STABILITY OF STANDARD COMPOSITIONS (PERCENTAGE OF ENZYMEACTIVITY REMAINING) UPON STORAGE AT 4° C. Formulation 1 2 4 5 6 No.¹Month Months Months Months Months 10 84 86 98  nd² 105 11 94 97 98 nd nd12 94 97 106 nd nd 13 86 93 93 nd nd 14 85 91 105 nd nd 15 89 89 98 ndnd 16 88 103 104 nd nd 17 83 94 91 nd nd 18 90 97 99 nd nd 19 95 59 ndnd 38 20 97 100 94 94 103 21 93 97 100 nd nd 22 100 109 35 nd nd 23 9497 89 nd 107 24 93 94 97 nd nd ¹Formulation numbers correspond to thosein Table 1. ²nd = not done

Several of the formulations were stable upon storage at 20-25° C., mostnotable formulation 20 which retained >70% of its enzymatic activityeven after storage for four months at this temperature. As describedearlier, formulation 19 had completely lost activity within the firstmonth, as determined by the PCR assay. Interestingly, however, asdetermined by the polymerase unit activity assay, formulation 19 wasstable for one month when stored at 4° C. but had lost substantialactivity by the second month of storage at this temperature (Table 4).Formulation 20, shown previously to be stable upon extended storage at20-25° C., was stable upon storage at 4° C. for at least six months.

Taken together, these results indicate that the compositions of thepresent invention are readily suitable for use in nucleic acidamplification reactions, and demonstrate extended stability upon storageat 20-25° C. or 4° C.

Example 3 Formulation and Stability of Large Sequence Compositions

For use in amplification and sequencing of nucleic acid fragments largerthan 5-6 kilobases, it has been suggested as described above that amixture of Taq and VENT™ or DEEPVENT™ polymerases (U.S. Pat. No.5,436,149; Barnes, Id), or of Tth and either Tli, Pyrococcus or Tma(U.S. Pat. No. 5,512,462), be used. Accordingly, Taq and DEEPVENT™ DNApolymerases were formulated, at activity ratios of 100:1 (for samples1-3) or 50:1 (for sample 4) into solutions containing the buffer salts,cofactors and detergents shown in Table 5. Each of these formulationswas adjusted to about pH 9.1, which is optimal for the activity ofDEEPVENT™ DNA polymerase (Bej and Mahbubani, Id). Samples were thenstored at about 20-25° C. or at 4° C. and assayed weekly for 12 weeks,and monthly thereafter, in stability assays in which a 20 kilobasetarget in 100 nanograms of human genomic template was amplified by PCR.Reaction mixtures included 10 picomoles of primer and were subjected to35 PCR cycles of 30 seconds at 94° C., 30 seconds at 62° C. and 1minute/kilobase at 68° C. Portions of the reaction were subjected toagarose gel electrophoresis and were visualized by ethidium bromidefluorescence under ultraviolet illumination, as shown above for FIGS. 1and 2. Results are summarized in Table 5.

TABLE 5 STABILITY OF LARGE SEQUENCE COMPOSITIONS Formulation Stabilityat: No. Formulation 20-25° C. 4° C. 1 66 mM Tris-SO₄ (pH 9.1) <12 1119.8 mM (NH₄)₂ SO₄ weeks months 2.2 mM MgSO₄ 22 units/ml Taq DNAPolymerase 0.22 units/ml DEEPVENT DNA Polymerase 0.11% Tween-20 0.011%NP-40 2 66 mM Tris-SO₄ (pH 9.1) <12 >11 19.8 mM (NH₄)₂ SO₄ weeks months2.2 mM MgSO₄ 24.42 units/ml Taq DNA Polymerase 0.242 units/ml DEEPVENTDNA Polymerase 0.066% Tween-20 0.066% NP-40 3 66 mM Tris-SO₄ (pH 9.1)nd¹ 11 19.8 mM (NH₄)₂ SO₄ months 2.2 mM MgSO₄ 22 units/ml Taq DNAPolymerase 0.22 units/ml DEEPVENT DNA Polymerase 0.01% Tween-20 0.01%NP-40 4 66 mM Tris-SO₄ (pH 9.1) nd¹ 11 19.8 mM (NH₄)₂ SO₄ months 2.2 mMMgSO₄ 22 units/ml Taq DNA Polymerase 0.44 units/ml DEEPVENT DNAPolymerase 0.01% Tween-20 0.01% NP-40 ¹nd = not done

Upon storage at ambient temperature (20°-25° C.), all of theformulations were stable for 6-12 weeks. Storage of these formulationsat 4° C. provided enhanced stability of over 11 months. Similar resultsmay be obtained with formulations in which Taq and Tne DNA polymeraseswere used in an activity ratio of 1:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:25,1:50, 1:100, 1:250, 1:500, 1:1000 or 1:2000. These results indicate thatthe large sequence compositions of the present invention are readilysuitable for use in amplification of nucleic acid sequences larger than5-6 kilobases and demonstrate extended stability upon storage at 20° to25° C., or at 4° C.

Example 4 Combinations of Thermus flavis (Tfl) DNA Polymerase andThermotoga neapolitana (Tne) DNA Polymerase

To examine other DNA polymerase compositions for their utility inamplification of nucleic acid molecules, a mixture of Tfl and Tne DNApolymerases, at a 1:1 ratio, was used to amplify the 2.7 kilobase Puc19plasmid. Amplification reactions were in a 50 μl final volume in buffercontaining 1 mM magnesium acetate. 80 pg of Puc19 linearized bytreatment with AdtII was used as the template, and was contacted with 1μl of enzyme mixture. PCR conditions were 1 min at 94° C., followed by35 cycles of 94° C. for 30 seconds/60° C. for 30 seconds/68° C. for 5minutes.

Upon analysis of the amplification products by gel electrophoresis, thiscomposition comprising Tfl and Tne DNA polymerases was found toefficiently amplify the 2.7 kilobase Puc19 plasmid. The efficiency ofamplification was comparable to amplification of Puc19 using 1 μl of TaqDNA polymerase.

Example 5 Amplification of Genomic DNA Using Tfl/Tne Compositions

Having demonstrated that compositions comprising Tfl and Tne DNApolymerases efficiently amplify plasmid-sized nucleic acid molecules,these compositions were examined for their ability to amplify nucleicacid molecules from genomic DNA templates. Six different primer setswere constructed (ranging in size from 0.25 to 4.1 kilobases) and usedto amplify the human β-globin gene from a genomic DNA template from theK562 human leukemia cell line. Each reaction was performed in a volumeof 50 μl comprising template at 40 ng/reaction, and Tfl/Tne mixture ateither 0.5 unit/reaction or 1 unit/reaction (Tfl and Tne at a 1:1 ratioin both mixtures). PCR conditions were 1 min at 94° C., followed by 35cycles of 94° C. for 30 seconds/55° C. for 30 second/68° C. for 5minutes.

Upon analysis of the amplification products by gel electrophoresis,efficient amplification was observed for all primers. The sizes of theamplification products produced using the different primers were 0.25kilobase, 0.7 kilobase, 1.1 kilobases, 2.0 kilobases and 4.1 kilobases.These results demonstrate that the Tfl/Tne compositions efficientlyamplify nucleic acid molecules derived from genomic DNA templates.

Example 6 Use of Various Enzyme Ratios in DNA Polymerase Compositions

To determine the efficacy of different enzyme ratios in nucleic acidamplification, a composition comprising a higher amount of Tne (3′ exo+)DNA polymerase than Tfl (3′ exo−) DNA polymerase was made and tested forits ability to amplify Puc19 and the β-globin gene. A 1:3 mixture ofTfl/Tne was made and used to amplify Puc19 under the conditionsdescribed in Example 4, and β-globin under the conditions described inExample 5.

Upon analysis of the amplification products by gel electrophoresis,efficient amplification was observed for both Puc19 and for β-globin(all template sizes). These results demonstrate that compositions inwhich a 3′ exo+ DNA polymerase (Tne) is present in higher quantity thana 3′ exo− DNA polymerase (Tfl) efficiently amplify nucleic acidmolecules derived from plasmid and genomic DNA templates.

Example 7 Amplification of Large Nucleic Acid Molecules Using DNAPolymerase Mixtures

Having demonstrated that compositions comprising mixtures of 3′ exo+ and3′ exo− DNA polymerases efficiently amplify plasmid-sized and smallgenomic nucleic acid molecules, such compositions were examined fortheir ability to amplify larger nucleic acid molecules. For thesestudies, mixtures of Taq (3′ exo+) DNA polymerase and Tma (3′ exo−) DNApolymerase (ULTma™; LTI, Rockville, Md.) were prepared. Two sets ofmixtures were prepared: one set contained 1 unit of Taq and varyingamounts of ULTma (0.3 unit, 0.6 unit, 0.8 unit or 1 unit), and the otherset contained no Taq and only varying amounts of ULTma (0.3 unit, 0.6unit, 0.8 unit or 1 unit). These compositions were used to amplify 80 mgof human genomic DNA using primers specific for a 7.5 kilobase region ofthe human β-globin gene. PCR conditions were 1 min at 94° C., followedby 35 cycles of 94° C. for 30 seconds/60° C. for 30 seconds/68° C. for 5minutes.

Upon analysis of the amplification products by gel electrophoresis, the7.5 kilobase β-globin fragment was found to be efficiently amplified byall compositions comprising both Taq and Tma DNA polymerase.Compositions comprising only Tma DNA polymerase, however, were unable toamplify this large fragment. In control experiments, compositionscomprising only Taq DNA polymerase were also unable to amplify thislarge fragment. These results demonstrate that compositions comprising amixture of 3′ exo+ and 3′ exo− DNA polymerases are useful in efficientlyamplifying large nucleic acid molecules, particularly in amplifyingnucleic acid molecules larger than about 7 kilobases in size.

Example 8 Amplification of Large Nucleic Acid Molecules Using VariousRatios of Taq and Tma DNA Polymerases

Having demonstrated that compositions comprising mixtures of 3′ exo+(Taq) and 3′ exo− (Tma) DNA polymerases efficiently amplify largenucleic acid molecules, compositions comprising these enzymes in variousratios were made and tested for their abilities to amplify the 7.5kilobase β-globin gene fragment from Example 7. Two sets of mixtureswere prepared: one set contained 1 unit of ULTma DNA polymerase anddifferent amounts (0.25, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 unit) of Taq DNApolymerase, and the other set contained 1 unit of Taq DNA polymerase anddifferent amounts (1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 3.0, 4.0 or 6.0units) of ULTma DNA polymerase. Amplification templates, primers andcycling conditions were as described for Example 7.

Upon analysis of the amplification products by gel electrophoresis,efficient amplification of the 7.5 kilobase β-globin gene was observedfor the first set of JO mixtures only in those compositions containing0.5 to 1.0 unit of Taq DNA polymerase and 1.0 unit of ULTma.Compositions in this set containing less than 0.5 unit of Taq DNApolymerase did not amplify this fragment. However, all enzyme mixturesin the other set, i.e., compositions comprising 1.0 unit of Taq DNApolymerase and 1.0 to 6.0 units of ULTma DNA polymerase, demonstratedefficient amplification of the 7.5 kilobase fragment. In separateexperiments, a 13.5 kilobase fragment of the β-globin gene wasefficiently amplified using mixtures containing a 1:1 or a 1:2 ratio ofTaq to ULTma. Together, these results indicate that compositions inwhich Tina DNA polymerase is present in equal or higher quantity thanTaq DNA polymerase efficiently amplify large nucleic acid molecules,particularly those that are larger than about 7-13 kilobases in size.

Example 9 Use of Taq and Tne DNA Polymerase Mixtures for Long PCR

To determine if other DNA polymerases could be used in compositions alsocomprising Taq DNA polymerase in amplification of large nucleic acidmolecules, various mixtures of Taq and Tne DNA polymerases were made.For these experiments, a Tne DNA polymerase deletion mutant (5′ exo−; 3′exo+) was mixed in amounts ranging from 0.05 to 2.0 units with 1 unit ofTaq DNA polymerase and used to amplify the 7.5 kilobase β-globinfragment under conditions described for Example 7.

Upon analysis of the amplification products by gel electrophoresis, allof the combinations of Tne and Taq DNA polymerases were found toefficiently amplify the 7.5 kilobase DNA fragment. These resultsindicate that compositions comprising combinations of Taq and Tne DNApolymerases are useful in amplifying large nucleic acid molecules,particularly those larger than about 7 kilobases in size.

Example 10 Preparation and Use of Compositions Comprising Anti-TaqAntibodies

To examine the stability of ready-to-use PCR reagents containinganti-Taq antibodies, Taq DNA polymerase was reacted with monoclonalantibody TP4-3 at ratios of 5:1, 2:1, 1:1 and 0:1 of antibody to Taq DNApolymerase. Binding of the antibody to Taq DNA polymerase inhibitedpolymerase activity of Taq almost completely at 5:1 and 2:1 ratios. The1:1 ratio of antibody to Taq resulted in partial inhibition ofpolymerase activity ranging from 54% to 83% of the control Taq DNApolymerase with no antibody.

The ready-to-use reaction mixtures were stored at 4° C. or −20° C. fordetermination of stability of Taq DNA polymerase as well as stability ofanti-Taq antibodies. Long-term storage of these mixtures showed noreduction in activity of Taq DNA polymerase or anti-Taq antibody after17 months at 4° C. or −20° C. (Table 6).

TABLE 6 Stability of Ready-to-Use PCR Reagents Ready-to-Use Ready-to-UseReagents Reagents Control Fresh Mixtures (4° C. Storage) (−20° C.Storage) Antibody:Taq Antibody Activity¹ Antibody Activity AntibodyActivity Ratio A B C D 14 months 17 months 14 months 17 months 5:1 97%98%   97% 97.9% 94.8% 97.5% 94.8% 98.5%   2:1 N.D.² 96.9%   96.4% N.D.N.D. N.D. 94.8% 98% 1:1 54% 79% 82.6%   68%   71%   65%   73% 67% 0:1(control)  0%  0%   0%   0%   0%   0%   0%  0% ¹Antibody activity isexpressed as % inhibition of DNA polymerization activity compared to noantibody (0:1 ratio) control. ²N.D. = not determined.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A stable composition comprising a mixture ofreagents at working concentrations, wherein said reagents are at leastone thermostable enzyme and at least one buffer salt.
 2. A stablecomposition for nucleic acid amplification comprising a mixture ofreagents, wherein said reagents are at least one thermostable DNApolymerase, at least one buffer salt and at least one deoxynucleosidetriphosphate.
 3. A stable composition for nucleic acid sequencingcomprising a mixture of reagents, wherein said reagents are at least onethermostable DNA polymerase, at least one deoxynucleoside triphosphate,at least one dideoxynucleoside triphosphate and at least one buffersalt.
 4. The stable composition of claim 2 or claim 3, wherein saidreagents are present at working concentrations.
 5. The composition ofclaim 2 or claim 3, wherein said thermostable DNA polymerase is selectedfrom the group of thermostable DNA polymerases consisting of a Taq DNApolymerase, a Tne DNA polymerase, a Tma DNA polymerase, and mutantsthereof.
 6. The composition of claim 2 or claim 3, wherein saidthermostable DNA polymerase is selected from the group of thermostableDNA polymerases consisting of a Pfu DNA polymerase, a Pwo DNApolymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, and mutantsthereof.
 7. The composition of claim 5, wherein said mixture furthercomprises DEEPVENT™ DNA polymerase or VENT™ DNA polymerase.
 8. Thecomposition of claim 5, wherein the concentration of Taq DNA polymeraseor mutant thereof is about 0.1 to 200 units per milliliter.
 9. Thecomposition of claim 8, wherein the concentration is about 20 units permilliliter.
 10. The composition of claim 5, wherein the concentration ofTne DNA polymerase or mutant thereof is about 0.1 to 200 units permilliliter.
 11. The composition of claim 10, wherein the concentrationis about 20 units per milliliter.
 12. The composition of claim 5,wherein the concentration of Tma DNA polymerase or mutant thereof isabout 0.1 to 200 units per milliliter.
 13. The composition of claim 12,wherein the concentration is about 20 units per milliliter.
 14. Thecomposition of claim 6, wherein the concentration of VENT™ DNApolymerase or mutant thereof is about 0.1 to 200 units per milliliter.15. The composition of claim 14, wherein the concentration is about 20units per milliliter.
 16. The composition of claim 6, wherein theconcentration of DEEPVENT™ DNA polymerase or mutant thereof is about 0.1to 200 units per milliliter.
 17. The composition of claim 16 wherein theconcentration is about 20 units per milliliter.
 18. The composition ofclaim 6, wherein the concentration of Pfu DNA polymerase or mutantthereof is about 0.1 to 200 units per milliliter.
 19. The composition ofclaim 18 wherein the concentration is about 20 units per milliliter. 20.The composition of claim 6, wherein the concentration of Pwo DNApolymerase or mutant thereof is about 0.1 to 200 units per milliliter.21. The composition of claim 20 wherein the concentration is about 20units per milliliter.
 22. The composition of claim 7, wherein theconcentration of DEEPVENT™ DNA polymerase or VENT DNA polymerase isabout 0.002 to 200 units per milliliter.
 23. The composition of claim22, wherein the concentration is about 0.40 units per milliliter. 24.The composition of claim 2 or claim 3, wherein said DNA polymeraseretains at least 90% of the enzymatic activity for at least four weekswhen stored at about 20° C. to 25° C.
 25. The composition of claim 5,wherein said DNA polymerase retains at least 90% of the enzymaticactivity for at least one year when stored at about 4° C.
 26. Thecomposition of claim 2 or claim 3, further comprising a magnesium salt.27. The composition of claim 2 or claim 3, further comprising at leastone nonionic detergent.
 28. The composition of claim 2 or claim 3,wherein the concentration of said deoxynucleoside triphosphate is about200 to about 300 micromolar.
 29. The composition of claim 3, wherein theconcentration of said dideoxynucleoside triphosphate is about 0.08 toabout 5 micromolar.
 30. A nucleic acid amplification kit comprising oneor more containers, wherein a first container contains a stablecomposition comprising a mixture of reagents, wherein said reagents areat least one thermostable DNA polymerase, at least one buffer salt, andat least one deoxynucleoside triphosphate.
 31. A nucleic acid sequencingkit comprising one or more containers, wherein a first containercontains a stable composition comprising a mixture of reagents, whereinsaid reagents are at least one thermostable DNA polymerase, at least onebuffer salt, at least one deoxynucleoside triphosphate and at least onedideoxynucleoside triphosphate.
 32. The kit of claim 30 or 31, whereinsaid reagents are present at working concentrations.
 33. A method ofamplifying a nucleic acid molecule comprising contacting said nucleicacid molecule with the composition of claim
 2. 34. A method ofamplifying a nucleic acid molecule comprising contacting said nucleicacid molecule with a composition selected from the group consisting of:a composition comprising a thermostable 3′ exo+ DNA polymerase and athermostable 3′ exo− DNA polymerase wherein the concentrations of said3′ exo+ DNA polymerase and of said 3′ exo− DNA polymerase are equal, anda composition comprising a thermostable 3′ exo+ DNA polymerase and athermostable 3′ exo− DNA polymerase wherein the concentration of said 3′exo+DNA polymerase is higher than the concentration of said 3′ exo− DNApolymerase.
 35. A method of sequencing a nucleic acid moleculecomprising contacting said nucleic acid molecule with the composition ofclaim
 3. 36. A method of sequencing a nucleic acid molecule comprisingcontacting said nucleic acid molecule with a composition selected fromthe group consisting of: a composition comprising a thermostable 3′ exo+DNA polymerase and a thermostable 3′ exo− DNA polymerase wherein theconcentrations of said 3′ exo+ DNA polymerase and of said 3′ exo− DNApolymerase are equal, and a composition comprising a thermostable 3′exo+ DNA polymerase and a thermostable 3′ exo− DNA polymerase whereinthe concentration of said 3′ exo+DNA polymerase is higher than theconcentration of said 3′ exo− DNA polymerase.
 37. The method of any oneof claims 33-36, wherein said nucleic acid molecule is larger than about4 kilobases in size.
 38. The method of claim 37, wherein said nucleicacid molecule is larger than about 7 kilobases in size.
 39. The methodof claim 38, wherein said nucleic acid molecule is larger than about 8kilobases in size.
 40. A nucleic acid molecule amplified by the methodof claim 33 or claim
 34. 41. The nucleic acid molecule of claim 40,wherein said nucleic acid molecule is larger than about 4 kilobases insize.
 42. The nucleic acid molecule of claim 41, wherein said nucleicacid molecule is larger than about 7 kilobases in size.
 43. The nucleicacid molecule of claim 41, wherein said nucleic acid molecule is largerthan about 8 kilobases in size.
 44. The composition of claim 1, furthercomprising at least one antibody that specifically binds to saidthermostable enzyme.
 45. The composition of claim 2 or claim 3, furthercomprising at least one antibody that specifically binds to saidthermostable enzyme.
 46. The kit of claim 30 or claim 31, wherein saidmixture of reagents further comprises at least one antibody thatspecifically binds to said thermostable DNA polymerase.
 47. The kit ofclaim 30 or claim 31, further comprising one or more additionalcontainers containing at least one antibody that specifically binds tosaid thermostable DNA polymerase.